The long-term goal of this project is to characterize the post-synaptic mechanisms regulating synaptic transmission, particularly those that occur during homeostatic plasticity. At central synapses, changes in expression of post-synaptic receptors are thought to occur during activity-dependent plasticity, including homeostatic plasticity. We developed C. elegans as a model to study trafficking of an ionotropic glutamate receptor (GluR) GLR-1 at central synapses. We showed that GLR-1 levels are up regulated during activity blockade, a model for homeostatic plasticity, that clathrin-mediated endocytosis is required for this compensation, and that GLR-1 is sorted into either of two post-endocytic trafficking pathways (recycling versus degradation). Ubiquitylation of GLR-1 promotes its trafficking into the degradation pathway whereas RAB-2 promotes sorting into the recycling pathway. At neuromuscular junctions (NMJs), retrograde signals from muscles regulate the growth, stability, and strength of their motor neuron inputs. We identified a novel retrograde signaling pathway whereby increased muscle activity induces a muscle transcription factor (MEF-2), and MEF-2 in turn induces a retrograde signal that decreases presynaptic release of neurotransmitter. Here, we propose three aims to characterize these two forms of homeostatic regulation. First, we will determine which Rab proteins regulate GLR-1 trafficking. Rab GTPases regulate specific steps in protein transport; consequently, Rab mutant phenotypes will allow us to experimentally define discrete steps in GLR-1 trafficking. We are particularly interested in endosomal Rabs, because changes in endosomal trafficking have been implicated in activity dependent plasticity. Second, we will determine how the MEF-2- dependent retrograde message is regulated by muscle activity. We will test whether changes in endogenous muscle activity alter MEF-2 transcriptional activity, whether this is mediated by activity-dependent dephosphorylation of MEF-2 by calcineurin, and if MEF-2 activity is specifically coupled to nicotinic acetylcholine receptors. Third, we will identify genes that act downstream of MEF-2, and are required for the MEF-2-dependent retrograde message. One such gene (aex-1) was identified in preliminary studies. In summary, our experiments will provide new insights into the specific post-synaptic mechanisms underlying two forms of homeostatic regulation. Given the strong conservation of these pathways across phylogeny, it is likely that our experiments will provide new insights into the general mechanisms underlying these fundamental aspects of synaptic cell biology. PUBLIC HEALTH RELEVANCE: This proposal describes a coherent set of genetic, biochemical, and biophysical experiments designed to characterize post-synaptic mechanisms regulating synaptic transmission, and in particular those underlying homeostatic plasticity. Homeostatic plasticity refers to a set of compensatory mechanisms whereby synaptic transmission is adjusted to compensate for long-term perturbations in activity. Homeostatic plasticity has been proposed to play a pivotal role in several aspects of circuit development and function. By examining mutant animals in which these compensatory mechanisms are inactivated, our experiments will allow us to critically evaluate these models.